Rebecca Y. Klinger

Blood vessels engineered from human cells

Tissue engineering has made considerable progress in the past decade, but advances have stopped short of clinical application for most tissues. We postulated that an obstacle in engineering human tissues is the limited replicative capacity of adult somatic cells. To test this hypothesis, the effectiveness of telomerase expression to extend cellular lifespan was assessed in a model of human vascular tissue engineering. Telomerase expression in vascular cells isolated from elderly patients enabled the successful culture of engineered autologous blood vessels. Engineered vessels may one day provide a source of bypass conduit for patients with atherosclerotic disease.

Cardiac fibroblast paracrine factors alter impulse conduction and ion channel expression of neonatal rat cardiomyocytes

AIMS The pathological proliferation of cardiac fibroblasts (CFs) in response to heart injury results in fibrosis, which correlates with arrhythmia generation and heart failure. Here we systematically examined the effect of fibroblast-derived paracrine factors on electrical propagation in cardiomyocytes. METHODS AND RESULTS Neonatal rat cardiac monolayers were exposed for 24 h to media conditioned by CFs. Optical mapping, sharp microelectrode recordings, quantitative RT-PCR, and immunostaining were used to assess the changes in the propagation and shape of the action potential and underlying changes in gene and protein expression. The fibroblast paracrine factors produced a 52% reduction in cardiac conduction velocity, a 217% prolongation of action potential duration, a 64% decrease of maximum capture rate, a 21% increase in membrane resting potential, and an 80% decrease of action potential upstroke velocity. These effects were dose dependent and partially reversible with removal of the conditioned media. No fibroblast proliferation, cardiomyocyte apoptosis, or decreased connexin-43 expression, phosphorylation, and function were found in conditioned cardiac cultures. In contrast, the expression of the fast sodium, inward rectifying potassium, and transient outward potassium channels were, respectively, reduced 3.8-, 6.6-fold, and to undetectable levels. The expression of beta-myosin heavy chain increased 17.4-fold. No electrophysiological changes were observed from media conditioned by CFs in the presence of cardiomyocytes. CONCLUSION Paracrine factors from neonatal CFs alone produced significant electrophysiological changes in neonatal rat cardiomyocytes resembling those found in several cardiac pathologies.

Structural coupling of cardiomyocytes and noncardiomyocytes: quantitative comparisons using a novel micropatterned cell pair assay.

Well-controlled studies of the structural and functional interactions between cardiomyocytes and other cells are essential for understanding heart pathophysiology and for the further development of safe and efficient cell therapies. We established a novel in vitro assay composed of a large number of individual micropatterned cell pairs with reproducible shape, size, and region of cell-cell contact. This assay was applied to quantify and compare the frequency of expression and distribution of electrical (connexin43) and mechanical (N-cadherin) coupling proteins in 5,000 cell pairs made of cardiomyocytes (CMs), cardiac fibroblasts (CFs), skeletal myoblasts (SKMs), and mesenchymal stem cells (MSCs). We found that for all cell pair types, side-side contacts between two cells formed 4.5-14.3 times more often than end-end contacts. Both connexin43 and N-cadherin were expressed in all homotypic CM pairs but in only 13.4-91.6% of pairs containing noncardiomyocytes, where expression was either junctional (at the site of cell-cell contact) or diffuse (inside the cytoplasm). CM expression was exclusively junctional in homotypic pairs but predominantly diffuse in heterotypic pairs. Noncardiomyocyte homotypic pairs exhibited diffuse expression 1.7-8.7 times more often than junctional expression, which was increased 2.6-4.4 times in heterotypic pairs. Junctional connexin43 and N-cadherin expression, respectively, were found in 38.6 +/- 7.3 and 39.6 +/- 6.2% of CM-MSC pairs, 21.9 +/- 5.0 and 13.6 +/- 1.9% of CM-SKM pairs, and in only 3.8-9.6% of CM-CF pairs. Measured frequencies of protein expression and distribution were stable for at least 4 days. Described studies in micropatterned cell pairs shed new light on cellular interactions relevant for cardiac function and cell therapies.

Hemodynamic impact of dexmedetomidine administration in 15,656 noncardiac surgical cases

STUDY OBJECTIVE To assess the hemodynamic impact of dexmedetomidine administration in a large cohort of patients undergoing routine noncardiac surgery. DESIGN Retrospective database analysis. SETTING Major academic medical center. MEASUREMENTS A valid electronic preoperative history and physical record and electronic perioperative anesthesia record of all adults undergoing noncardiothoracic procedures of > 60 minutes duration between January 2007 and September 2008 were reviewed. The primary composite endpoint was systolic blood pressure < 80 mmHg for > 5 minutes, heart rate < 40 bpm for > 5 minutes, or administration of vasoconstrictors (> 500 μg of phenylephrine by bolus or infusion or any epinephrine) or atropine intraoperatively. MAIN RESULTS A total of 15,656 cases, of whom 2,688 (17%) received dexmedetomidine preoperatively or intraoperatively and 12,968 (83%) did not receive dexmedetomidine, was identified. A significantly higher percentage of patients in the dexmedetomidine group met the composite endpoint criteria (27% vs 19%, P < 0.0001). However, there was no significant difference in the overall incidence of intraoperative hypotension (5.3% dexmedetomidine, 6% no dexmedetomidine) or bradycardia (0.4% in both groups). Dexmedetomidine patients received more phenylephrine or atropine (23% vs 15%, P < 0.0001). CONCLUSIONS In a large cohort of routine clinical practice cases, dexmedetomidine administration was not associated with more hypotension or bradycardia.

Cardiac cell therapy in vitro: reproducible assays for comparing the efficacy of different donor cells [Cellular/Tissue Engineering]

Cardiovascular disease remains a pervasive and significant clinical problem, making it the target of extensive research for novel therapeutic strategies. In this report, we focus on describing the methods for the fabrication of cardiac strands with donor inserts and the application of this assay to test the ability of a few selected donor cell types to bridge and propagate electrical impulses from one cardiomyocyte area to another. We showed a set of reproducible, well-controlled in vitro assays that allow for the comparative and quantitative studies of cardiomyocyte and donor cell interactions on several functional levels. Cellular therapies are emerging rapidly as one of the more promising methodologies to repair injured myocardium.

Conduction block in micropatterned cardiomyocyte cultures replicating the structure of ventricular cross-sections

AIMS Structural and functional heterogeneities in cardiac tissue have been implicated in conduction block and arrhythmogenesis. However, the propensity of specific sites within the heart to initiate conduction block has not been systematically explored. We utilized cardiomyocyte cultures replicating the realistic, magnetic resonance imaging-measured tissue boundaries and fibre directions of ventricular cross-sections to investigate their roles in the development of conduction block. METHODS AND RESULTS The Sprague-Dawley neonatal rat cardiomyocytes were micropatterned to obtain cultures with realistic ventricular tissue boundaries and either random or realistic fibre directions. Rapid pacing was applied at multiple sites, with action potential propagation optically mapped. Excitation either failed at the stimulus site or conduction block developed remotely, often initiating reentry. The incidence of conduction block in isotropic monolayers (0% of cultures) increased with the inclusion of realistic tissue boundaries (17%) and further with realistic fibre directions (34%). Conduction block incidence was stimulus site-dependent and highest (77%) with rapid pacing from the right ventricular (RV) free wall. Furthermore, conduction block occurred exclusively at the insertion of the RV free wall into the septum, where structure-mediated current source-load mismatches acutely reduced wavefront and waveback velocity. Tissue boundaries and sharp gradients in fibre direction uniquely determined the evolution, shape, and position of conduction block lines. CONCLUSION Our study suggests that specific micro- and macrostructural features of the ventricle determine the incidence and spatiotemporal characteristics of conduction block, independent of spatial heterogeneities in ion channel expression.

A Multicenter Pilot Study Assessing Regional Cerebral Oxygen Desaturation Frequency During Cardiopulmonary Bypass and Responsiveness to an Intervention Algorithm.

BACKGROUND:The purpose of this multicenter pilot study was to: (1) determine the frequency of regional cerebral oxygen saturation (rScO2) desaturations during cardiac surgery involving cardiopulmonary bypass (CPB); (2) evaluate the accuracy of clinician-identified rScO2 desaturations compared with those recorded continuously during surgery by the near-infrared spectroscopy (NIRS) monitor; and (3) assess the effectiveness of an intervention algorithm for reversing rScO2 desaturations. METHODS:Two hundred thirty-five patients undergoing coronary artery bypass graft and/or valvular surgery were enrolled at 8 US centers in this prospective observational study. NIRS (Invos™ 5100C; Covidien) was used to monitor rScO2 during surgery. The frequency and magnitude of rScO2 decrements >20% from preanesthesia baseline were documented, and the efficacy of a standard treatment algorithm for correcting rScO2 was determined. The data from the NIRS monitor were downloaded at the conclusion of surgery and sent to the coordinating center where the number of clinician-identified rScO2 desaturation events was compared with the number detected by the NIRS monitor. RESULTS:The average rScO2 obtained at baseline (mean ± SD, 61% ± 11%; 99% confidence interval, 57%–65%) and during CPB (62% ± 14%; 57%–67%) was not different. However, rScO2 after separation from CPB (56% ± 11%; 53%–60%) was lower than measurements at baseline and during CPB (P < 0.001). During CPB, rScO2 desaturations occurred in 61% (99% confidence interval, 50%–75%) of patients. The area under the curve for product of magnitude and duration of the rScO2 was (mean ± SD, 145.2; 384.8% × min). Clinicians identified all patients with an rScO2 desaturation but identified only 340 (89.5%) of the 380 total desaturation events. Of the 340 clinician-identified rScO2 desaturation events, 115 resolved with usual clinical care before implementation of the treatment algorithm. For the remaining 225 events, the treatment algorithm resulted in resolution of the rScO2 desaturation in all but 18 patients. CONCLUSIONS:This multicenter pilot study found that 50% to 75% of patients undergoing cardiac surgery experience one or more rScO2 desaturations during CPB. Nearly 10% of desaturation events were not identified by clinicians, suggesting that appropriate alarming systems should be adopted to alert clinicians of such events. The intervention algorithm was effective in reversing clinically identified rScO2 desaturations in the majority of events.

Intraoperative Magnesium Administration Does Not Reduce Postoperative Atrial Fibrillation After Cardiac Surgery.

BACKGROUND: Hypomagnesemia has been associated with an increased risk of postoperative atrial fibrillation (POAF). Although previous studies have suggested a beneficial effect of magnesium (Mg) therapy, almost all of these are limited by small sample size and relatively low Mg dose. We hypothesized that high-dose Mg decreases the occurrence of new-onset POAF, and we tested this hypothesis by using data from a prospective trial that assessed the effect of Mg on cognitive outcomes in patients undergoing cardiac surgery. METHODS: A total of 389 patients undergoing cardiac surgery were enrolled in this double-blind, placebo-controlled trial. Subjects were randomized to receive Mg as a 50-mg/kg bolus immediately after induction of anesthesia followed by another 50 mg/kg as an infusion given over 3 hours (total dose, 100 mg/kg) or placebo. We tested the effect of Mg therapy on POAF with logistic regression, adjusting for the risk of atrial fibrillation (AF) by using the Multicenter Study of Perioperative Ischemia risk index for Atrial Fibrillation after Cardiac Surgery. RESULTS: Among the 363 patients analyzed, after we excluded patients with chronic or acute preoperative AF (placebo: n = 177; Mg: n = 186), the incidence of new-onset POAF was 42.5% (95% confidence interval [CI], 35%–50%) in the Mg group compared with 37.9% (95% CI, 31%–45%) in the placebo group (P = 0.40). The 95% CI for this absolute risk difference of 4.6% is −5.5% to 14.7%. The time to onset of POAF also was identical between the groups, and no significant effect of Mg was found in logistic regression analysis after we adjusted for AF risk (odds ratio, 1.09; 95% CI, 0.69–1.72; P = 0.73). CONCLUSIONS: High-dose intraoperative Mg therapy did not decrease the incidence of new-onset POAF after cardiac surgery.

Adverse outcomes associated with postoperative atrial arrhythmias after lung transplantation: A meta-analysis and systematic review of the literature

Postoperative atrial arrhythmias (AAs) are common after lung transplantation, but studies are mixed regarding their impact on outcomes. We therefore performed this systematic review and meta‐analysis to determine whether AAs after lung transplantation impede postoperative recovery.

Cortical β-amyloid levels and neurocognitive performance after cardiac surgery.

Introduction Neurological and neurocognitive dysfunction occurs frequently in the large number of increasingly elderly patients undergoing cardiac surgery every year. Perioperative cognitive deficits have been shown to persist after discharge and up to several years after surgery. More importantly, perioperative cognitive decline is predictive of long-term cognitive dysfunction, reduced quality of life and increased mortality. The proposed mechanisms to explain the cognitive decline associated with cardiac surgery include the neurotoxic accumulation of β-amyloid. This study will be the first to provide molecular imaging to assess the relationship between neocortical β-amyloid deposition and postoperative cognitive dysfunction. Methods and analysis 40 patients providing informed consent for participation in this Institutional Review Board-approved study and undergoing cardiac (coronary artery bypass graft (CABG), valve or CABG+valve) surgery with cardiopulmonary bypass will be enrolled based on defined inclusion and exclusion criteria. At 6 weeks after surgery, participants will undergo 18F-florbetapir positron emission tomography imaging to assess neocortical β-amyloid burden along with a standard neurocognitive battery and blood testing for apolipoprotein E ε-4 genotype. Results The results will be compared to those of 40 elderly controls and 40 elderly patients with mild cognitive impairment who have previously completed 18F-florbetapir imaging. Ethics and dissemination This study has been approved by the Duke University Institutional Review Board. The results will provide novel mechanistic insights into postoperative cognitive dysfunction that will inform future studies into potential treatments or preventative therapies of long-term cognitive decline after cardiac surgery.